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PubMed1991

The Aeromonas hydrophila cphA gene: molecular heterogeneity among class B metallo-beta-lactamases.

An Aeromonas hydrophila gene, named cphA, coding for a carbapenem-hydrolyzing metallo-beta-lactamase, was cloned in Escherichia coli by screening an Aeromonas genomic library for clones able to grow on imipenem-containing medium. From sequencing data, the cloned cphA gene appeared able to code for a polypeptide of 254 amino acids whose sequence includes a potential N-terminal leader sequence for targeting the protein to the periplasmic space. These data were in agreement with the molecular mass of the original Aeromonas enzyme and of the recombinant enzyme produced in E. coli, evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of crude beta-lactamase preparations followed by renaturation treatment for proteins separated in the gel and localization of protein bands showing carbapenem-hydrolyzing beta-lactamase activity by a modified iodometric technique. The deduced amino acid sequence of the CphA enzyme showed regions of partial homology with both the beta-lactamase II of Bacillus cereus and the CfiA beta-lactamase of Bacteroides fragilis. Sequence homologies were more pronounced in the regions encompassing the amino acid residues known in the enzyme of B. cereus to function as ligand-binding residues for the metal cofactor. The CphA enzyme, however, appeared to share a lower degree of similarity with the two other enzymes, which, in turn, seemed more closely related to each other. These results, therefore, suggest the existence of at least two molecular subclasses within molecular class B metallo-beta-lactamases.

Aeromonas↗
PubMed1997

The disulfide bond in the Aeromonas hydrophila lipase/acyltransferase stabilizes the structure but is not required for secretion or activity.

Vibrio and Aeromonas spp. secrete an unusual 35-kDa lipase that shares several properties with mammalian lecithin-cholesterol acyltransferase. The Aeromonas hydrophila lipase contains two cysteine residues that form an intramolecular disulfide bridge. Here we show that changing either of the cysteines to serine does not reduce enzymatic activity, indicating that the disulfide bond is not required for correct folding. However, when either of the cysteines is replaced, the enzyme is more readily denatured by urea and more sensitive to degradation by trypsin than is the wild-type enzyme, evidence that the bridge has an important role in stabilizing the protein's structure. The two mutant proteins with serine-for-cysteine replacements were secreted by Aeromonas salmonicida containing the cloned genes, although the levels of both in the culture supernatants were lower than the level of the wild-type enzyme. When the general secretory pathway was blocked with carbonyl cyanide chlorophenylhydrazone, the cell-associated pools of the mutant enzymes appeared to be degraded, whereas the wild-type pool remained stable. We conclude that reduced extracellular levels of the mutant proteins are the result of their increased sensitivities to proteases encountered inside the cell during export.

Acyltransferases↗
PubMed1997

Identification and molecular characterization of two tandemly located flagellin genes from Aeromonas salmonicida A449.

Two tandemly located flagellin genes, flaA and flaB, with 79% nucleotide sequence identity were identified in Aeromonas salmonicida A449. The fla genes are conserved in typical and atypical strains of A. salmonicida, and they display significant divergence at the nucleotide level from the fla genes of the motile species Aeromonas hydrophila and Aeromonas veronii biotype sobria. flaA and flaB encode unprocessed flagellins with predicted Mrs of 32,351 and 32,056, respectively. When cloned under the control of the Ptac promoter, flaB was highly expressed when induced in Escherichia coli DH5alpha, and the FlaB protein was detectable even in the uninduced state. In flaA clones containing intact upstream sequence, FlaA was barely detectable when uninduced and poorly expressed on induction. The A. salmonicida flagellins are antigenically cross-reactive with the A. hydrophila TF7 flagellin(s) and evolutionarily closely related to the flagellins of Pseudomonas aeruginosa and Vibrio anguillarum. Electron microscopy showed that A. salmonicida A449 expresses unsheathed polar flagella at an extremely low frequency under normal laboratory growth conditions, suggesting the presence of a full complement of genes whose products are required to make flagella; e.g., immediately downstream of flaA and flaB are open reading frames encoding FlaG and FlaH homologs.

Aeromonas↗
PubMed1969

Aldohexuronic acid catabolism by a soil Aeromonas.

Bacteria which utilize mannuronic acid as an energy source were isolated from nature. One of the organisms, identified as a member of the genus Aeromonas, used glucuronate, galacturonate, and mannuronate as the sole source of carbon and energy. Glucuronate- and galacturonate-grown resting cells oxidized both glucuronate and galacturonate rapidly, but mannuronate slowly. Mannuronate-grown cells oxidized all three rapidly, with the rate of mannuronate utilization somewhat lower. Cell-free extracts from glucuronate-, galacturonate-, and mannuronate-grown Aeromonas C11-2B contained glucuronate and galacturonate isomerases, fructuronate, tagaturonate, and mannuronate reductases, and mannonate and altronate dehydratases, with the exception of glucuronate-grown cells which lacked altronate dehydratase. Thus, the pathway for glucuronate and galacturonate catabolism for Aeromonas was identical to Escherichia coli. Glucuronate and galacturonate were isomerized to d-fructuronate and d-tagaturonate which were then reduced by reduced nicotinamide adenine dinucleotide to d-mannonate and d-altronate, respectively. The hexonic acids were dehydrated to 2-keto-3-deoxy gluconate which was phosphorylated by adenosine triphosphate to 2-keto-3-deoxy-6-phospho gluconate. The latter was then cleaved to pyruvate and glyceraldehyde-3-phosphate. Mannuronate was reduced directly to d-mannonate by a reduced nicotinamide adenine dinucleotide phosphate-linked oxidoreductase. d-Mannonate was then further broken down as in the glucuronate pathway. The mannuronate reducing enzyme, for which the name d-mannonate:nicotinamide adenine dinucleotide (phosphate) oxidoreductase (d-mannuronate-forming) was proposed, was shown to be distinct from altronate and mannoate oxidoreductases. This is the first report of a bacterial oxidoreductase which reduces an aldohexuronic acid to a hexonic acid. The enzyme should prove to be a useful analytical tool for determining mannuronate in the presence of other uronic acids.

Aeromonas↗
PubMed1982

Biochemical characteristics of enterotoxigenic Aeromonas spp.

Biotypes of Aeromonas spp. correlated well with enterotoxin production in a study of 174 strains. Using biochemical characteristics determined by conventional methods and multitest systems, we correctly classified 93% of the strains with regard to enterotoxin production. Most of the enterotoxigenic strains were Voges-Proskauer (VP) positive and did not hydrolyze arabinose, but VP-positive strains which hydrolyzed arabinose were mainly non-enterotoxigenic. Aeromonas punctata subsp. caviae, which is VP negative and does not oxidize gluconate or produce gas from glucose, was non-enterotoxigenic. Although the number of other VP-negative strains was small, most were enterotoxigenic. Discrimination was improved so that 97% of the strains were correctly classified if the hemolysin assay was used either for all strains or for only the VP-positive, arabinose-positive and VP-negative, non-A. punctata subsp. caviae strains. Because the proposed classification system does not require facilities for carrying out in vivo assays such as suckling mouse or ileal loop methods, the identification of enterotoxigenic Aeromonas strains should be possible in diagnostic laboratories and will facilitate epidemiological studies of the role of these organisms in acute diarrhea.

Aeromonas↗
PubMed1983

Evaluation of differential and selective media for isolation of Aeromonas and Plesiomonas spp. from human feces.

We studied nine solid and two liquid media for their suitability to select Aeromonas and Plesiomonas spp. from human stools, using artificially contaminated samples as well as 254 samples from outpatients with and without diarrhea. Media with optimal sensitivity and specificity for Aeromonas spp. were alkaline peptone-water, Trypticase soy broth with ampicillin, inositol-brilliant green-bile salts agar, dextrin-fuchsin-sulfite agar, xylose-sodium desoxycholate-citrate agar, and Pril-xylose-ampicillin agar. For Plesiomonas sp., alkaline peptone-water and inositol-brilliant green-bile salts agar were optimal. Four strains of Aeromonas spp. were detected in patient samples with these media.

Aeromonas↗
PubMed1983

Correlation of enterotoxicity with biotype in Aeromonas spp.

Enterotoxin production correlated with biotype in a study of 686 strains of Aeromonas spp. from Indonesia, Thailand, the United States, and Western Australia. Most strains were isolated from feces but nonfecal human isolates and environmental strains were also included. More than 80% of Voges-Proskauer (VP)-positive strains, classified as A. hydrophila, were enterotoxigenic in the suckling mouse assay as were 90% of VP-positive, arabinose-negative strains. An association between positive VP, arabinose fermentation, and failure to produce enterotoxins was found only with environmental strains. VP-negative strains which did not oxidize gluconate or produce gas from glucose were classified as A. punctata subsp. caviae. Only 2 of the 286 strains produced enterotoxins, and both were from Indonesian fecal samples. There were few remaining VP-negative strains, classified as A. punctata subsp. punctata and, of these, about half were enterotoxigenic. Regardless of source and species, 97% of Aeromonas spp. were correctly classified in relation to enterotoxin production with a hemolysin assay. A combination of biochemical testing and hemolysin assay should be suitable for diagnostic laboratories to identify enterotoxigenic Aeromonas spp. which, in children, are associated with diarrhea, unlike non-enterotoxigenic strains.

Aeromonas↗
PubMed1984

Enterotoxin production in relation to taxonomic grouping and source of isolation of Aeromonas species.

A total of 19 of 20 (95%) strains of Aeromonas hydrophila biovar hydrophila and 16 of 17 (94%) strains of Aeromonas sobria isolated from a variety of clinical and environmental sources were found to be enterotoxin positive. Only 2 of 18 (11%) A. hydrophila biovar anaerogenes and 2 of 13 (15%) unidentified Aeromonas strains from a similar variety of sources produced enterotoxin. No association was apparent between the source of isolation, in particular diarrheal stools, and enterotoxigenicity; 41% of the isolates from diarrheal stools were enterotoxin negative. A strong correlation was noted between ability to produce enterotoxin and positive results in six characters: lysine decarboxylase and Voges-Proskauer reactions, production of gas from glucose, gluconate oxidation, xanthine hydrolysis, and hemolysis of human erythrocytes. In the majority of cases (35 of 39 strains), enterotoxigenicity was detected using cell-free filtrates of brain heart infusion broth cultures grown at 36 degrees C for 15; however, the other four positive isolates were detected after growth in the same broth at 30 degrees C or in Casamino Acids-yeast extract broth at 30 or 37 degrees C. It is recommended that for enterotoxin tests, strains should be grown in both media at both temperatures. The infant mouse test was found to be a simple and reliable method for detection of the enterotoxin. The toxin proved to be heat labile and not neutralized by cholera antitoxin.

Aeromonas↗
PubMed1986

Growth of Aeromonas species on enteric agars.

The efficacy of eight routine enteric agars for supporting the growth of 32 strains of Aeromonas spp. (17 A. hydrophila strains, 8 A. sobria strains, and 7 A. caviae strains) was investigated. The plating efficiency of Aeromonas spp. on these media varied greatly (range, 0 to 100%), as did their colony size when compared with that on noninhibitory medium (5% sheep blood agar). Plating efficiency on seven of these eight media appeared to be strain- and not species dependent. Overall, eosin-methylene blue and Hektoen enteric agars showed low plating efficiencies for A. hydrophila, whereas both A. sobria and A. caviae were severely inhibited on brilliant green agar. When all these species are considered collectively, deoxycholate, MacConkey, and xylose lysine deoxycholate appeared to be the most satisfactory routine agars for Aeromonas spp. recovery when used in conjunction with blood agar.

Aeromonas↗
PubMed1986

Production of "Asao toxin" by Aeromonas strains isolated from feces and drinking water.

Cultures of Aeromonas species were tested for production of a toxin recently purified by Asao et al. (T. Asao, Y. Kinoshita, S. Kozaki, T. Uemura, and G. Sukaguchi, Infect. Immun. 46:122-127, 1984) and described as a hemolysin with enterotoxic and cytotoxic activity. The toxin was produced by only 63% of Aeromonas sobria strains and by 93% of Aeromonas hydrophila strains. Also, 54% of A. hydrophila strains produced another cytotoxic entity.

Aeromonas↗
PubMed1986

Virulence characteristics of Aeromonas spp. in relation to source and biotype.

The significance of Aeromonas spp. as potential water-borne enteric pathogens in Tasmania, Australia, an area with a mild climate and comparatively low year-round water temperatures, was investigated in view of the reported marked peak of Aeromonas-associated gastroenteritis in the summer and the apparent influence of temperature on levels of potentially pathogenic species in water supplies. Biochemical characteristics and virulence-associated properties--exotoxin production (hemolysin, enterotoxin), ability to grow at 43 degrees C, and possession of pili--were determined for 105 Tasmanian isolates of Aeromonas spp.; 43 isolates were from clinical specimens (greater than 75% diarrhea associated) and 62 were from water. Current classification schemes were evaluated for these isolates. A. sobria comprised 35% of the clinical isolates and 16% of the water isolates, A. hydrophila comprised 56 and 79%, and A. caviae comprised 9 and 5%. A total of 42% of the clinical isolates and 15% of the environmental isolates were enterotoxigenic (by the suckling mouse assay); these levels were significantly lower than those found in warmer environments. The majority (74%) of enterotoxigenic isolates were A. sobria. Enterotoxin-producing isolates possessed three or more of the following properties. They were Voges-Proskauer positive, did not hydrolyze arabinose, were positive for lysine decarboxylase, were able to grow at 43 degrees C, and produced large amounts of hemolysin (titer, greater than 128). Thus, the biochemical scheme proposed by Burke et al. (V. Burke, J. Robinson, H.M. Atkinson, and M. Gracey, J. Clin. Microbiol. 15:48-52, 1982) for identifying enterotoxigenic isolates appears to have widespread applicability. Environmental enterotoxigenic isolates possessed numerous pili, but these appeared to be lost once infection was established, as a similar isolates from patients with diarrhea were poorly piliated.

Aeromonas↗
PubMed1987

Congo red uptake by motile Aeromonas species.

Virulence of several species of enteropathogenic bacteria has been correlated with the ability of isolates to take up the dye Congo red. To determine whether Congo red uptake might be a useful marker for virulence of motile Aeromonas species, we examined 50 strains of diverse clinical origin on a medium containing 50 micrograms of Congo red per ml. All of the strains took up the dye to various degrees. For most strains, uptake was greatest at 37 degrees C and least at 22 degrees C. Production of acetyl methyl carbinol (Voges-Proskauer test) or lysine decarboxylase has been reported by some investigators to be a virulence marker for Aeromonas species. Congo red uptake did not correlate with either acetyl methyl carbinol or lysine decarboxylase production in our study. These data suggest that Congo red uptake may not be a useful marker for virulence of motile Aeromonas species.

Aeromonas↗
PubMed1988

Correlation of the suicide phenomenon in Aeromonas species with virulence and enteropathogenicity.

Certain strains of mesophilic aeromonads (Aeromonas hydrophila, A. sorbria, and A. caviae), when grown in broth containing 0.5% glucose, undergo growth inhibition concomitant with acetate accumulation. Because these strains are nonviable after 24 h, this phenomenon is termed suicide. We investigated suicidal strains of Aeromonas species as a means of understanding animal virulence and enteropathogenicity. To assess virulence, batches of five white mice were inoculated intraperitoneally with 10(7) cells (washed) of suicidal and nonsuicidal strains of A. hydrophila and A. sobria and suicidal strains of A. caviae. The three nonsuicidal strains of A. sobria tested showed lethality as early as 12 h and were uniformly fatal within 36 h postinoculation. After 36 h, the three suicidal strains killed only 1 of 15 mice inoculated. Four A. hydrophila strains tested which showed the suicide phenomenon at 37 degrees C were variably lethal (40 to 100%). None of three suicidal strains of A. caviae were lethal. Enteropathogenicity was studied by orally inoculating three white mice each with the same Aeromonas strains (10(8) cells, in skim milk) and assessing diarrhea and intestinal fluid accumulation. Diarrhea and fluid accumulation were present in all mice inoculated with two nonsuicidal strains of A. sobria and in 4 of 12 mice given four suicidal strains of A. hydrophila. Two suicidal strains each of A. sorbria and A. caviae failed to elicit any gastrointestinal disturbances. These data suggest that the suicide phenomenon may explain strain-specific (A. sobria and A. hydrophila) and species-specific (A. caviae) virulence and enteropathogenicity.

Aeromonas↗
PubMed1990

Microbiologic and clinical evidence supporting the role of Aeromonas caviae as a pediatric enteric pathogen.

Aeromonas caviae was recovered as the sole potential enteric pathogen from the stools of 14 of 17 symptomatic children (10 younger than 1 year of age) while Aeromonas hydrophila, Aeromonas sobria, and Plesiomonas shigelloides were isolated once each. The infants from whom A. caviae was isolated all presented with a watery diarrhea lasting 1 to 3 weeks. None of these infants was breast-fed, and all had a stool pH of greater than 7.5. All of the A. caviae isolates, including a reference strain (ATCC 15468), adhered to HEp-2 cells, and preliminary data showed that they produced a cytotoxin as well. Because A. caviae can survive at an elevated pH, as found in the gastrointestinal tract of formula-fed infants, and because of the adherence and cytotoxin production capabilities of the species, it should be regarded as an enteric pathogen in pediatric patients and most probably among adults as well.

Aeromonas↗
PubMed1990

Isolation and identification of autoagglutinating serogroup O:11 Aeromonas strains in the clinical laboratory.

We evaluated the extent to which serogroup O:11 Aeromonas strains could be recovered from both clinical and environmental specimens and the cultural parameters that affected the phenotypic marker (autoagglutination) associated with this group. Of over 200 Aeromonas strains screened, serogroup O:11 was identified only among the phenospecies A. hydrophila and A. sobria and was associated with clinical isolates more frequently than with environmental strains. Blood and wound isolates accounted for almost 50% of all O:11 strains identified. The autoagglutination phenotype associated with O:11 strains could be detected in most commercial liquid media, under a wide range of growth temperatures, and within 15 min of incubation at 100 degrees C. The results suggest that clinical laboratories can recognize this important group of Aeromonas strains by two simple tests.

Aeromonas↗
PubMed1990

Transient intestinal colonization by multiple phenotypes of Aeromonas species during the first week of life.

The intestinal colonization rate of Aeromonas spp. was determined for 52 cesarean-born Peruvian neonates. Rectal swabs were obtained daily from newborns during their postdelivery hospitalization (mean = 5.5 days), and the gross appearances of their feces (blind determinations) were recorded. Aeromonas spp. were recovered from rectal swabs of 12 of 52 (23.1%) infants during their first week of life; the isolates were obtained from 5 of 9 (55.6%) infants with at least one stool with a watery consistency and from 7 of 43 (16.3%) neonates with no watery stools (P = 0.022). None of the infected infants became clinically ill. No other commonly recognized enteropathogens were detected in watery stools. An environmental survey indicated that hospital water was the probable source of infection. These and other data indicated that Aeromonas colonization occurs transiently at a very early age in Peruvian neonates and that in some instances, initial infection may be followed several days later by one or more watery stools of normal volume.

Aeromonas↗
PubMed1998

Inducible beta-lactam resistance in Aeromonas hydrophila: therapeutic challenge for antimicrobial therapy.

Despite the abundant amount of knowledge about inducible chromosomally mediated beta-lactamases among Aeromonas species, extended-spectrum beta-lactam-resistant A. hydrophila strains selected in clinical practice were rarely reported. In the present study, two strains of A. hydrophila, A136 and A139, with markedly different susceptibilities to extended-spectrum cephalosporins were isolated from blood and the tip segment of an arterial catheter of a burn patient. Another strain (A136m) was selected in vitro by culturing A136 in a subinhibitory concentration of cefotaxime, the beta-lactam agent administered for the treatment of Aeromonas bacteremia in this patient. Typing studies by arbitrarily primed PCR and pulsed-field gel electrophoresis indicated a clonal relationship among strains A136, A136m, and A139. These strains were identified to be of DNA hybridization group 1. Wild-type strain A136 was resistant only to ampicillin and cephamycins, but A136m and A139 were highly resistant to the expanded- and broad-spectrum cephalosporins. The presence of increased beta-lactamase activity in A139 suggests that A139 is a derepressed mutant which overexpresses beta-lactamases. These results call attention to the use of beta-lactam agents for the treatment of invasive Aeromonas infections.

Aeromonas hydrophila↗